Plasmids are normally circular, extrachromosomal DNA molecules that replicate autonomously within the cells of host organisms. The cells of many unicellular organisms, including some bacteria, contain naturally occurring wild-type plasmids that contribute various functions to the host cells such as antibiotic resistance and fertility. These wild-type plasmids and derivatives of them are the basic tools of recombinant DNA technology, providing vehicles for the transformation of the cells of host organisms with foreign DNA sequences which code for production, within the transformed cells, of corresponding foreign products. As used herein, “foreign products” means DNA, RNA and/or polypeptides that are foreign to the host cells including but not limited to DNA sequences encoding foreign genes, RNA molecules transcribed from inserted additional foreign DNA sequences, and polypeptides encoded by foreign genes.
Recombinant plasmids, similar to the parent plasmids from which they are derived, are capable of autonomous replication within host cells, and on replication, reproduce not only the DNA sequences of the parent plasmid but also the inserted additional DNA sequences, including the foreign genes. During polypeptide synthesis, transcription and translation of the DNA sequences of the recombinant plasmids carried within transformed host cells give rise to the synthesis of foreign RNA and polypeptides.
One factor which affects the yield of synthesized foreign product is the number of copies of the foreign gene which are present within the transformed cells, i.e. the copy number at which the recombinant plasmid is maintained within the host cells, this being defined normally as the number of copies of the plasmid per host genome. Generally speaking, the higher the copy number of the recombinant plasmid the greater is the yield of foreign product. Both low-copy-number plasmids, usually maintained within host cells at about 1-10 copies per genome, and high copy number plasmids, usually maintained at from 11 up to several hundred copies per genome, are known. The copy number of a given wild-type replicon is controlled by DNA sequences surrounding and including a DNA sequence that defines the origin of replication (“ori”). Thus, hereinafter we refer to high copy number and low copy number ori's.
High copy number plasmids have been used in recombinant systems with a view to obtaining good yields of foreign products. This can lead to undesirable results, however, since many such high copy number plasmids tend not to be maintained stably within transformed cells and may be lost from the cells before they can be grown to sufficient levels to permit bulk production of foreign products. For example, the foreign product may be toxic and/or inhibit propagation of the transformed cells, or the high copy number plasmids themselves may be inherently unstable or recombine with DNA sequences in other copies of the recombinant plasmid that are present in the same cell.
It is known that the copy numbers of some plasmids can be amplified above normal levels by inhibition of protein synthesis, such as, by addition of protein synthesis inhibitors such as chloramphenicol to the fermentation medium. However, protein synthesis is required for production of most gene products, and therefore the inhibitor must be removed before synthesis of foreign products can take place. The removal of inhibitor requires complicated manipulations and is not always possible.
Other solutions have also been proposed to overcome the problem of stable maintenance of high copy number plasmids in host cells. For example, in U.S. Pat. Nos. 4,487,835; 4,495,287; and 4,499,189, incorporated herein by reference, Uhlin et al. disclosed the use of mutant plasmids having a temperature-dependent plasmid copy number pattern such that the plasmid shows a controlled constant plasmid copy number when host bacteria carrying the plasmid are cultivated at one temperature and a much higher or totally uncontrolled copy number when the host bacteria carrying the plasmid are grown at a different temperature. Thus, cells may be propagated to desired production size culture at one temperature at which the plasmid replicates at low copy number and at which its gene products do not significantly inhibit cell growth. The temperature may then be altered, greatly increasing the plasmid copy number and also the corresponding production of gene products. However, temperature-dependent copy number may be limited to particular mutant plasmids, which may or may not contain suitable restriction enzyme cloning sites for a particular foreign DNA sequence. Also, introduction of copy number temperature dependence may introduce a source of instability into the plasmid, and these mutant plasmids may be unstable or subject to loss when cells carrying them are propagated over a prolonged period of time. Another disadvantage of this approach is the fact that higher temperatures may have a negative impact on protein stability.
In U.S. Pat. No. 5,015,573, Yarranton et al., incorporated herein by reference, disclosed a new class of vectors to solve the problem of stable low-copy maintenance of the vector while permitting replication at high copy number under a different set of conditions to produce a high yield of gene product. These vectors had two origins of replication. When propagated under a first set of conditions, replication takes place using the first on and results in a low copy number and stable inheritance of the vector or recombinant vector containing foreign DNA. Then, when propagated under a second set of conditions, replication takes place using a second, controllable ori, which is under the control of an inducible promoter. In one embodiment of this invention, the natural promoter which promotes transcription of the RNA species (RNAII or a similar species) that provides a primer for initiation of DNA replication by formation of a complex at or near the origin of replication is replaced by a controllable promoter. If a heterologous cloned gene is also under the control of a controllable promoter, both replication and expression of the gene are controllable from their respective promoters.
Like the invention disclosed by Yarranton, et al. U.S. Pat. No. 6,472,177 of Szybalski et al. and U.S. Pat. No. 5,874,259 of Szybalski, both incorporated herein by reference, disclosed compositions and methods for controlling copy number of a plasmid, including a BAC plasmid, wherein the plasmid contains two origins of replication. According to U.S. Pat. No. 6,472,177, which primarily discusses compositions and methods for dual control of both replication and transcription, “the conditional origin is provided in addition to a origin of replication that maintains the vector at a single copy per cell,” and “the conditional on could be any on that functions in the host cell and is normally inactive until exposed to the replication-inducing agent.” Thus, neither Yarranton nor Szybalski disclose compositions or methods for controlling plasmid copy number by controlling replication from a single ori.
U.S. Pat. No. 6,165,749 of Sagawa et al., incorporated herein by reference, also discloses vectors and methods for controlling the expression of a desired gene by a combination of two control mechanisms, i.e., by control of the copy number of the vector containing the gene and by control of transcription of the gene via an inducible promoter attached to an RNA polymerase gene. Use of these two control mechanisms enabled successful expression of a restriction enzyme that was toxic to the host cell when expressed without control of copy number of the vector containing the gene. In the case of this invention, the control of plasmid copy number was obtained by placing the RNAII gene, a replication pre-primer for initiation of replication from the plasmid ori, under an inducible promoter. Induction of the gene for RNAII resulted in an increase in copy number of plasmids containing the ori.
While the compositions and methods disclosed in the art provide solutions for controlling copy number of recombinant plasmids for particular applications, they suffer from certain disadvantages. All of the methods are limited to vectors having particular additional genetic elements, genes or other modifications. For example, the method of Uhlin et al. requires the use of a vector containing a particular mutation that causes temperature-sensitivity. The methods of Yarranton et al. and of Szybalski et al. require the use of vectors with two origins of replication, which increases the size of the vector and in most cases will limit the number and kind of restriction enzyme sites available for cloning of foreign genes. The method of Sagawa is limited to vectors that contain particular inducible RNAII-encoding DNA sequences for high-copy replication of the vector and, in most cases, an inducible RNA polymerase gene for transcription of a foreign gene that is cloned in the vector.
What is needed in the art are host cells and methods that enable copy-number control of replication of a broad range of widely-available plasmids from ori's that are capable of low-copy replication under one set of conditions and of high-copy replication under another set of conditions.
What is needed are host cells and methods that do not require modification or genetic engineering of the plasmid in order to control the copy number of the plasmid or of recombinant clones made using the plasmid.
Preferably, what is needed are host cells and methods for easily maintaining commonly-used plasmids and plasmid clones at low copy number for stable maintenance of clones and minimal loss of cloned DNA sequences that would be toxic or detrimental to the host cell at high copy number, and yet, which permit the plasmid and plasmid clones to be induced to high copy number in a tightly-controlled manner by means of simple reagents or conditions in order to obtain larger amounts and therefore, also, a higher purity of foreign products for the desired application.
Most preferably, what is needed are host cells and methods for inducible control of replication and copy number of a broad range of plasmids that have ori's with antisense-mediated replication control mechanisms, such as plasmids having ori's of the types contained in ColE1- and R1-type plasmids.